The goals of this project are to understand recombinational DNA repair, particularly the mechanisms of the central recombinases involved in this process and their regulation. Homologous genetic recombination is at once (a) a key DNA repair process, (b) one of the important cancer avoidance pathways in higher eukaryotes, and (c) one of the primary paths to the productive alteration/engineering of cellular genomes. This competing proposal is focused on the bacterial RecA protein, proteins that regulate or augment RecA function, and proteins that function in closely related pathways. We will also explore specific applications of these proteins in biotechnology. There are four specific aims. Aim 1 is focused on RecA itself. We will generate RecA variants with enhanced functionality, which may eventually find application in biotechnology. Aims 2 and 3 focus on proteins that have a major role in maintaining genome stability, but address molecular functions that have been largely overlooked. The subject of Aim2 is the protein MgsA (maintenance of genome stability A). MgsA has close homologues in all organisms from bacteria to humans, but its function has been enigmatic. We have found that it opens up DNA ends, presumably to facilitate the productive loading of key helicases that function in DNA replication. Aim 3 addresses a previously uncharacterized protein called RadD. This protein may play a role in removing barriers such as RNA polymerase from damaged DNA, clearing the way for DNA repair processes. Finally, Aim 4 will continue our efforts to elucidate the role of RecA protein in the function of the mutagenic DNA polymerase V. We have made much progress in the past 4 years on our understanding of this unusual DNA polymerase, and are now down to mapping the molecular interaction between RecA and the polymerase subunits. The RecA variants generated in Aim 1 may have some practical application as the subjects of new studies to explore RecA structure/function and as reagents in RecA applications in forensic science and genome engineering.